Exponential base-greater-than-2 nucleic acid amplification

ABSTRACT

Described herein are methods and compositions that provide highly efficient nucleic acid amplification. In some embodiments, this allows a greater than 2-fold increase of amplification product for each amplification cycle and therefore increased sensitivity and speed over conventional PCR.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. 371 National Phase Application ofPCT/US2015/065890, filed Dec. 15, 2015, which claims the benefit of U.S.provisional application No. 62/092,102, filed Dec. 15, 2014, which ishereby incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable.

FIELD

The methods and compositions described herein relate generally to thearea of nucleic acid amplification. In particular, described herein aremethods and compositions for increasing amplification efficiency.

BACKGROUND

A wide variety of nucleic acid amplification methods are available, andmany have been employed in the implementation of sensitive diagnosticassays based on nucleic acid detection. Polymerase chain reaction (PCR)remains the most widely used DNA amplification and quantitation method.Nested PCR, a two-stage PCR, is used to increase the specificity andsensitivity of the PCR (U.S. Pat. No. 4,683,195). Nested primers for usein the PCR amplification are oligonucleotides having sequencecomplementary to a region on a target sequence between reverse andforward primer targeting sites. However, PCR in general has severallimitations. PCR amplification can only achieve less than two foldincrease of the amount of target sequence at each cycle. It is stillrelatively slow. In addition, the sensitivity of this method istypically limited, making it difficult to detect target that may bepresent at only a few molecules in a single reaction.

SUMMARY

Described herein are methods and compositions based on the use of novelprimers (e.g., novel inner primers) designed to so that the outer primerbinding site is maintained in the amplicons produced upon amplification.

Provided herein are the following embodiments: Embodiment 1 A nucleicacid primer set for amplifying a target nucleic acid in a sample,wherein the target nucleic acid includes a first template strand and,optionally, a second template strand, wherein the second template strandis complementary to the first template strand, the primer set includingoligonucleotides in the form of, or capable of forming, at least twofirst primers capable of hybridizing to the first template strand,wherein the at least two first primers include a first outer primer anda first inner primer,

the first outer primer including a primer sequence a that specificallyhybridizes to first template strand sequence a′; and

the first inner primer including a single-stranded primer sequence bthat specifically hybridizes to first template strand sequence b′,wherein b′ is adjacent to, and 5′ of, a′, and wherein single-strandedprimer sequence b is linked at its 5′ end to a first strand of adouble-stranded primer sequence including:

a primer sequence a adjacent to, and 5′ of, single-stranded primersequence b; and

a clamp sequence c adjacent to, and 5′ of, primer sequence a, whereinclamp sequence c is not complementary to a first strand templatesequence d′, which is adjacent to, and 3′ of, first strand templatesequence a′.

Embodiment 2: The primer set of embodiment 1, wherein the primer setadditionally includes at least one second primer capable of specificallyhybridizing to the second template strand.

Embodiment 3: A method for amplifying a target nucleic acid in a sample,wherein the target nucleic acid includes a first template strand and,optionally, a second template strand, wherein the second template strandis complementary to the first template strand, the method including:

(a) contacting the sample with:

(i) at least two first primers capable of hybridizing to the firsttemplate strand, wherein the at least two first primers comprise a firstouter primer and a first inner primer,

-   -   the first outer primer including a primer sequence a that        specifically hybridizes to first template strand sequence a′;        and    -   the first inner primer including a single-stranded primer        sequence b that specifically hybridizes to first template strand        sequence b′, wherein b′ is adjacent to, and 5′ of, a′, and        wherein single-stranded primer sequence b is linked at its 5′        end to a first strand of a double-stranded primer sequence        including:        -   a primer sequence a adjacent to, and 5′ of, single-stranded            primer sequence b; and        -   a clamp sequence c adjacent to, and 5′ of, primer sequence            a, wherein clamp sequence c is not complementary to a first            strand template sequence d′, which is adjacent to, and 3′            of, first strand template sequence a′; and

(ii) at least one second primer capable of specifically hybridizing tothe second template strand, wherein the contacting is carried out underconditions wherein the primers anneal to their template strands, ifpresent; and

(b) amplifying the target nucleic acid, if present, using a DNApolymerase lacking 5′-3′ exonuclease activity, under conditions wherestrand displacement occurs, to produce amplicons that comprise sequenceextending from template sequence a′ to the binding site for the secondprimer.

Embodiment 4: The primer set or method of any preceding embodiment,wherein the DNA polymerase includes strand displacement activity.

Embodiment 5: The primer set or method of any preceding embodiment,wherein the T_(m) of combined sequence c-a, in double-stranded form, isgreater than that of combined sequence a-b, in double stranded form.

Embodiment 6: The primer set or method of any preceding embodiment,wherein combined sequence c-a is more GC-rich than combined sequencea-b, and/or contains more stabilizing bases.

Embodiment 7: The method of embodiments 3-6, wherein said amplifyingamplifies the target nucleic acid at the rate of up to3^(number of cycles) during the exponential phase of PCR.

Embodiment 8: The method of embodiments 3-7, wherein said amplifyingpermits detection of a single copy nucleic acid in a biological samplewithin about 12%-42% fewer amplification cycles than would be requiredfor said detection using only a single forward and a single reverseprimer.

Embodiment 9: The primer set of embodiments 1, 2, 5, or 6 or the methodof embodiments 3-8, wherein the second primer includes oligonucleotidesin the form of, or capable of forming, at least two second primerscapable of hybridizing to the second template strand, wherein the atleast two second primers comprise a second outer primer and a secondinner primer,

the second outer primer including a primer sequence e that specificallyhybridizes to second template strand sequence e′;

and the second inner primer including a single-stranded primer sequencef that specifically hybridizes to second template strand sequence f′,wherein f′ is adjacent to, and 5′ of, e′, and wherein single-strandedprimer sequence f is linked at its 5′ end to a first strand of adouble-stranded primer sequence including:

-   -   a primer sequence e adjacent to, and 5′ of, single-stranded        primer sequence f; and    -   a clamp sequence g adjacent to, and 5- of, primer sequence e,        wherein clamp sequence g is not complementary to second strand        template sequence h′, which is adjacent to, and 3′, of second        strand template sequence e′.

Embodiment 10: The primer set or method of embodiment 9, wherein theT_(m) of combined sequence g-e, in double-stranded form is greater thanthat of combined sequence e-f, in double-stranded form.

Embodiment 11: The primer set or method of embodiments 9 or 10, whereincombined sequence g-e is more GC-rich than combined sequence e-f, and/orcontains more stabilizing bases.

Embodiment 12: The primer set or method of embodiments 9-11, whereinsaid amplifying amplifies the target nucleic acid at the rate of up to6^(number of cycles) during the exponential phase of PCR.

Embodiment 13: The primer set or method of embodiments 9-12, whereinsaid amplifying permits detection of a single copy nucleic acid in abiological sample within about 36%-66% fewer amplification cycles thanwould be required for said detection using only a single forward and asingle reverse primer.

Embodiment 14:The primer set or method of any preceding embodiment,wherein clamp sequence(s) c and g, if present, is/are not capable ofbeing copied during amplification.

Embodiment 15: The primer set or method of embodiment 14, wherein clampsequence(s) c and/or g, if present, comprise(s) 2′-O-methyl RNA.

Embodiment 16: The primer set or method of any preceding embodiment,wherein the double-stranded primer sequence of the first inner primerand/or the second inner primer, if present, does not include a hairpinsequence.

Embodiment 17: The primer set or method of embodiments 3-15, wherein thedouble-stranded primer sequence of the first inner primer includes ahairpin sequence in which clamp sequence c is linked to complementarysequence c′ and/or the double-stranded primer sequence of the secondinner primer, if present, includes a hairpin sequence in which clampsequence g is linked to complementary sequence g′.

Embodiment 18: A nucleic acid primer set for amplifying a target nucleicacid in a sample, wherein the target nucleic acid includes a firsttemplate strand and, optionally, a second template strand, wherein thesecond template strand is complementary to the first template strand,the primer set including oligonucleotides in the form of, or capable offorming, at least three first primers capable of hybridizing to thefirst template strand, wherein the at least three first primers comprisea first outer primer, a first intermediate primer, and a first innerprimer,

-   -   the first outer primer including a primer sequence d that        specifically hybridizes to first template strand sequence d′;    -   the first intermediate primer including a primer sequence a that        specifically hybridizes to first template strand sequence a′,        wherein a′ is adjacent to, and 5′ of, d′, and wherein        single-stranded primer sequence a is linked at its 5′ end to a        first strand of a double-stranded primer sequence including:        -   a primer sequence d adjacent to, and 5′ of, single-stranded            primer sequence a; and        -   a clamp sequence c1 adjacent to, and 5- of, primer sequence            d, wherein clamp sequence c1 is not complementary to a first            strand template sequence i′, which is adjacent to, and 3′            of, first strand template sequence d′; and    -   the first inner primer including a single-stranded primer        sequence b that specifically hybridizes to first template strand        sequence b′, wherein b′ is adjacent to, and 5′ of, a′, and        wherein single-stranded primer sequence b is linked at its 5′        end to a first strand of a double-stranded primer sequence        including:        -   a primer sequence a adjacent to, and 5′ of, single-stranded            primer sequence b;        -   a primer sequence d adjacent to, and 5′ of, primer sequence            a; and        -   a clamp sequence c2 adjacent to, and 5′ of, primer sequence            d, wherein clamp sequence c2 is not complementary to first            strand template sequence i′.

Embodiment 19: The primer set of embodiment 18, wherein the primer setadditionally includes at least one second primer capable of specificallyhybridizing to the second template strand.

Embodiment 20: A method for amplifying a target nucleic acid in asample, wherein the target nucleic acid includes a first template strandand, optionally, a second template strand, wherein the second templatestrand is complementary to the first template strand, the methodincluding:

(a) contacting the sample with:

-   -   (i) at least three first primers capable of hybridizing to the        first template strand, wherein the at least three first primers        comprise a first outer primer, a first intermediate primer, and        a first inner primer,        -   the first outer primer including a primer sequence d that            specifically hybridizes to first template strand sequence            d′;        -   the first intermediate primer including a primer sequence a            that specifically hybridizes to first template strand            sequence a′, wherein a′ is adjacent to, and 5′ of, d′, and            wherein single-stranded primer sequence a is linked at its            5′ end to a first strand of a double-stranded primer            sequence including:            -   a primer sequence d adjacent to, and 5′ of,                single-stranded primer sequence a; and            -   a clamp sequence c1 adjacent to, and 5- of, primer                sequence d, wherein clamp sequence c1 is not                complementary to a first strand template sequence i′,                which is adjacent to, and 3′ of, first strand template                sequence d′; and            -   the first inner primer including a single-stranded                primer sequence b that specifically hybridizes to first                template strand sequence b′, wherein b′ is adjacent to,                and 5′ of, a′, and wherein single-stranded primer                sequence b is linked at its 5′ end to a first strand of                a double-stranded primer sequence including:            -   a primer sequence a adjacent to, and 5′ of,                single-stranded primer sequence b;            -   a primer sequence d adjacent to, and 5′ of, primer                sequence a; and            -   a clamp sequence c2 adjacent to, and 5′ of, primer                sequence d, wherein clamp sequence c2 is not                complementary to first strand template sequence i′; and    -   (ii) at least one second primer capable of specifically        hybridizing to the second template strand, wherein the        contacting is carried out under conditions wherein the primers        anneal to their template strands, if present; and

(b) amplifying the target nucleic acid, if present, using a DNApolymerase lacking 5′-3′ exonuclease activity, under conditions wherestrand displacement occurs, to produce amplicons that comprise sequenceextending from template sequence a′ to the binding site for the secondprimer.

Embodiment 21: The primer set or method of embodiments 18-20, whereinthe DNA polymerase includes strand displacement activity.

Embodiment 22: The primer set or method of embodiments 18-21, wherein g1has a different sequence than g2.

Embodiment 23: The primer set or method of embodiments 18-22, whereinthe T_(m) of combined sequence c1-d, in double-stranded form, is greaterthan that of combined sequence d-a, in double-stranded form, and theT_(m) of combined sequence c2-d-a, in double-stranded form, is greaterthan that of combined sequence d-a-b, in double-stranded form.

Embodiment 24: The primer set or method of embodiments 18-23, whereincombined sequence c1-d is more GC-rich than combined sequence d-a,and/or contains more stabilizing bases, and combined sequence c2-d-a ismore GC-rich than combined sequence d-a-b, and/or contains morestabilizing bases than combined sequence d-a-b.

Embodiment 25: The method of embodiments 20-24, wherein said amplifyingamplifies the target nucleic acid at the rate of up to4^(number of cycles) during the exponential phase of PCR.

Embodiment 26: The method of embodiments 20-25, wherein said amplifyingpermits detection of a single copy nucleic acid in a biological samplewithin about 25%-55% fewer amplification cycles than would be requiredfor said detection using only a single forward and a single reverseprimer.

Embodiment 27: The primer set or method of embodiments 18-26, whereinthe second primer includes oligonucleotides in the form of, or capableof forming, at least three second primers capable of hybridizing to thesecond template strand, wherein the at least three second primerscomprise a second outer primer, a second intermediate primer, and asecond inner primer,

the second outer primer including a primer sequence h that specificallyhybridizes to second template strand sequence h′;

the second intermediate primer including a single-stranded primersequence e that specifically hybridizes to second template strandsequence e′, wherein e′ is adjacent to, and 5′ of, h′, and whereinsingle-stranded primer sequence e is linked at its 5′ end to a firststrand of a double-stranded primer sequence including:

-   -   a primer sequence h adjacent to, and 5′ of, single-stranded        primer sequence e; and    -   a clamp sequence g1 adjacent to, and 5′ of, primer sequence h,        wherein clamp sequence g1 is not complementary to a second        strand template sequence j′, which is adjacent to, and 3′, of        second strand template sequence h′; and

the second inner primer including a single-stranded primer sequence fthat specifically hybridizes to first template strand sequence f′,wherein f′ is adjacent to, and 5′ of, e′, and wherein single-strandedprimer sequence f is linked at its 5′ end to a first strand of adouble-stranded primer sequence including:

-   -   a primer sequence e adjacent to, and 5′ of, single-stranded        primer sequence f;    -   a primer sequence h adjacent to, and 5′ of, primer sequence e;        and    -   a clamp sequence g2 adjacent to, and 5′ of, primer sequence h,        wherein clamp sequence c2 is not complementary to first strand        template sequence j′.

Embodiment 28: The primer set or method of embodiment 27, wherein theT_(m) of combined sequence g1-h, in double-stranded form, is greaterthan that of combined sequence h-e, in double-stranded form, and theT_(m) of combined sequence g2-h-e, in double-stranded form, is greaterthan that of combined sequence h-e-f, in double-stranded form.

Embodiment 29: The primer set or method of embodiments 27 or 28, whereincombined sequence g1-h is more GC-rich than combined sequence h-e,and/or contains more stabilizing bases, and combined sequence g2-h-e ismore GC-rich than combined sequence h-e-f, and/or contains morestabilizing bases than combined sequence h-e-f.

Embodiment 30: The method of embodiments 27-29, wherein said amplifyingamplifies the target nucleic acid at the rate of up to8^(number of cycles) during the exponential phase of PCR.

Embodiment 31: The method of embodiments 27-30, wherein said amplifyingpermits detection of a single copy nucleic acid in a biological samplewithin about 42%-72% fewer amplification cycles than would be requiredfor said detection using only a single forward and a single reverseprimer.

Embodiment 32: The primer set or method of embodiments 18-31, whereinclamp sequences c1 and c2, and g1 and g2, if present, are not capable ofbeing copied during amplification.

Embodiment 33: The primer set or method of embodiment 32, wherein clampsequences c1 and c2, and g1 and g2, if present, include 2′-O-methyl RNA.

Embodiment 34: The primer set or method of embodiments 20-33, wherein:the double-stranded primer sequence of the first inner primer and thefirst intermediate primer; and/or the second inner primer and the secondintermediate primer, if present, does/do not comprise a hairpinsequence.

Embodiment 35: The primer set or method of embodiments 20-33, wherein:the double-stranded primer sequence of the first inner primer includes ahairpin sequence in which clamp sequence c2 is linked to complementarysequence c2′; and/or the double-stranded primer sequence of the firstintermediate primer includes a hairpin sequence in which clamp sequencec1 is linked to complementary sequence c1′; and/or the double-strandedprimer sequence of the second inner primer, if present, includes ahairpin sequence in which clamp sequence g2 is linked to complementarysequence g2′; and/or the double-stranded primer sequence of the secondintermediate primer, if present, includes a hairpin sequence in whichclamp sequence g1 is linked to complementary sequence g1′.

Embodiment 36: The method of embodiments 3-17 or 20-35, wherein theamplification includes PCR.

Embodiment 37: The method of embodiments 3-17 or 20-36, wherein the DNApolymerase includes strand displacement activity and is thermostable.

Embodiment 38: The method of embodiments 3-17 or 20-37, wherein themethod includes detecting, and optionally quantifying, the targetnucleic acid.

Embodiment 39: The method of embodiments 3-17 or 20-38, wherein thesample consists of nucleic acids from a single cell.

Embodiment 40: The primer set or method of any of embodiments 1-6,wherein combined sequence a-b contains more destabilizing bases thancombined sequence c-a.

Embodiment 41: The primer set or method of embodiments 9-11, whereincombined sequence e-f contains more destabilizing bases than combinedsequence g-e.

Embodiment 42: The primer set or method of embodiments 18-24, whereincombined sequence d-a contains more destabilizing bases than combinedsequence c1-d, and/or combined sequence d-a-b contains moredestabilizing bases than combined sequence c2-d-a.

Embodiment 43: The primer set or method of embodiments 27-29, whereincombined sequence h-e contains more destabilizing bases than combinedsequence g1-h, and/or combined sequence h-e-f contains moredestabilizing bases than combined sequence g2-h-e.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A schematic showing fully nested PCR being carried out on adouble-stranded DNA template. The flanking primers are as described forFIG. 2 and FIG. 3.

FIG. 2: A schematic showing an illustrative two-primer set hybridized toone end of a target nucleotide sequence. This set can be, e.g., aforward primer set. Different segments of primer sequence are shown (a,b, c); complementary sequences are indicated as (a′, b′, c′). Templatesequences are indicated 3′-5′ as d′, a′, and b′. The outer primer (a) issingle-stranded. The inner primer has a single stranded portion (b) anda double-stranded portion (a-c).

FIG. 3: A schematic showing an illustrative two-primer set hybridized tothe opposite end of a target nucleotide sequence from that shown in FIG.2. This set can be, e.g., a reverse primer set. Different segments ofprimer sequence are shown (e, f, g); complementary sequences areindicated as (e′, f′, g′). Template sequences are indicated 3′-5′ as h′,e′, and f′. The outer primer (e) is single-stranded. The inner primerhas a single stranded portion (f) and a double-stranded portion (a-g).

FIG. 4: A schematic showing an illustrative three-primer set hybridizedto one end of a target nucleotide sequence. This set can be, e.g., aforward primer set. Different segments of primer sequence are shown (a,b, c1, c2, d); complementary sequences are indicated as (a′, b′, c1′,c2′, d′). Template sequences are indicated 3′-5′ as i′, d′, a′, and b′.The outer primer (d) is single-stranded. The intermediate primer has asingle stranded portion (a) and a double-stranded portion (d-c1). Theinner primer has a single stranded portion (b) and a double-strandedportion (a-d-c2).

FIG. 5: A schematic showing an illustrative three-primer set hybridizedto the opposite end of a target nucleotide sequence from that shown inFIG. 4. This set can be, e.g., a reverse primer set. Different segmentsof primer sequence are shown (e, f, g1, g2, h); complementary sequencesare indicated as (e′, f′, g1′, g2′, h′). Template sequences areindicated 3′-5′ as j′, h′, e′, and f. The outer primer (d) issingle-stranded. The intermediate primer has a single stranded portion(e) and a double-stranded portion (h-g1). The inner primer has a singlestranded portion (f) and a double-stranded portion (e-h-g2).

FIG. 6A-B: A schematic drawing showing two alternative structures forthe illustrated primer having a clamp sequence when the primer isallowed to hybridize with template. A fluorescent quencher (Q) ispresent in the primer in a position where it quenches a correspondingfluorescent label (F) in the template strand. In Example 1, anexperiment was performed in which the Tm was measured of the primer anda complimentary target sequence with and without the clamp present. (A)The structure formed if the T_(m) of combined sequence c-a, indouble-stranded form, is greater than that of combined sequence a-b, indouble stranded form. (B) The structure formed if the T_(m) of combinedsequence c-a, in double-stranded form, is less than that of combinedsequence a-b, in double stranded form.

FIG. 7A-B: (A) A schematic showing an illustrative two-primer set inwhich a fluorescent quencher (Q) is present in the inner primer in aposition where it quenches a corresponding fluorescent label (F) in thetemplate strand. (B) Fluorescence intensity as a function of time fromthe primer extension reaction of Example 2. The three rising traces areseparate reactions with slightly different clamps; the flat trace iswithout the outer (flanking), displacing primer present.

DETAILED DESCRIPTION

Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

The term “nucleic acid” refers to a nucleotide polymer, and unlessotherwise limited, includes known analogs of natural nucleotides thatcan function in a similar manner (e.g., hybridize) to naturallyoccurring nucleotides.

The term nucleic acid includes any form of DNA or RNA, including, forexample, genomic DNA; complementary DNA (cDNA), which is a DNArepresentation of mRNA, usually obtained by reverse transcription ofmessenger RNA (mRNA) or by amplification; DNA molecules producedsynthetically or by amplification; mRNA; and non-coding RNA.

The term nucleic acid encompasses double- or triple-stranded nucleicacid complexes, as well as single-stranded molecules. In double- ortriple-stranded nucleic acid complexes, the nucleic acid strands neednot be coextensive (i.e, a double-stranded nucleic acid need not bedouble-stranded along the entire length of both strands).

The term nucleic acid also encompasses any chemical modificationsthereof, such as by methylation and/or by capping. Nucleic acidmodifications can include addition of chemical groups that incorporateadditional charge, polarizability, hydrogen bonding, electrostaticinteraction, and functionality to the individual nucleic acid bases orto the nucleic acid as a whole. Such modifications may include basemodifications such as 2′-position sugar modifications, 5-positionpyrimidine modifications, 8-position purine modifications, modificationsat cytosine exocyclic amines, substitutions of 5-bromo-uracil,sugar-phosphate backbone modifications, unusual base pairingcombinations such as the isobases isocytidine and isoguanidine, and thelike.

More particularly, in some embodiments, nucleic acids, can includepolydeoxyribonucleotides (containing 2-deoxy-D-ribose),polyribonucleotides (containing D-ribose), and any other type of nucleicacid that is an N- or C-glycoside of a purine or pyrimidine base, aswell as other polymers containing nonnucleotidic backbones, for example,polyamide (e.g., peptide nucleic acids (PNAs)) and polymorpholinopolymers (commercially available from the Anti-Virals, Inc., Corvallis,Oreg., as Neugene), and other synthetic sequence-specific nucleic acidpolymers providing that the polymers contain nucleobases in aconfiguration which allows for base pairing and base stacking, such asis found in DNA and RNA. The term nucleic acid also encompasses lockednucleic acids (LNAs), which are described in U.S. Pat. Nos. 6,794,499,6,670,461, 6,262,490, and 6,770,748, which are incorporated herein byreference in their entirety for their disclosure of LNAs.

The nucleic acid(s) can be derived from a completely chemical synthesisprocess, such as a solid phase-mediated chemical synthesis, from abiological source, such as through isolation from any species thatproduces nucleic acid, or from processes that involve the manipulationof nucleic acids by molecular biology tools, such as DNA replication,PCR amplification, reverse transcription, or from a combination of thoseprocesses.

As used herein, the term “complementary” refers to the capacity forprecise pairing between two nucleotides; i.e., if a nucleotide at agiven position of a nucleic acid is capable of hydrogen bonding with anucleotide of another nucleic acid to form a canonical base pair, thenthe two nucleic acids are considered to be complementary to one anotherat that position. Complementarity between two single-stranded nucleicacid molecules may be “partial,” in which only some of the nucleotidesbind, or it may be complete when total complementarity exists betweenthe single-stranded molecules. The degree of complementarity betweennucleic acid strands has significant effects on the efficiency andstrength of hybridization between nucleic acid strands.

“Specific hybridization” refers to the binding of a nucleic acid to atarget nucleotide sequence in the absence of substantial binding toother nucleotide sequences present in the hybridization mixture underdefined stringency conditions. Those of skill in the art recognize thatrelaxing the stringency of the hybridization conditions allows sequencemismatches to be tolerated.

In some embodiments, hybridizations are carried out under stringenthybridization conditions. The phrase “stringent hybridizationconditions” generally refers to a temperature in a range from about 5°C. to about 20° C. or 25° C. below than the melting temperature (T_(m))for a specific sequence at a defined ionic strength and pH. As usedherein, the T_(m) is the temperature at which a population ofdouble-stranded nucleic acid molecules becomes half-dissociated intosingle strands. Methods for calculating the T_(m) of nucleic acids arewell known in the art (see, e.g., Berger and Kimmel (1987) METHODS INENZYMOLOGY, VOL. 152: GUIDE TO MOLECULAR CLONING TECHNIQUES, San Diego:Academic Press, Inc. and Sambrook et al. (1989) MOLECULAR CLONING: ALABORATORY MANUAL, 2ND ED., VOLS. 1-3, Cold Spring Harbor Laboratory),both incorporated herein by reference for their descriptions ofstringent hybridization conditions). As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (see, e.g., Anderson and Young, Quantitative FilterHybridization in NUCLEIC ACID HYBRIDIZATION (1985)). The meltingtemperature of a hybrid (and thus the conditions for stringenthybridization) is affected by various factors such as the length andnature (DNA, RNA, base composition) of the primer or probe and nature ofthe target nucleic acid (DNA, RNA, base composition, present in solutionor immobilized, and the like), as well as the concentration of salts andother components (e.g., the presence or absence of formamide, dextransulfate, polyethylene glycol). The effects of these factors are wellknown and are discussed in standard references in the art. Illustrativestringent conditions suitable for achieving specific hybridization ofmost sequences are: a temperature of at least about 60° C. and a saltconcentration of about 0.2 molar at pH7. Tm calculation foroligonuclotide sequences based on nearest-neighbors thermodynamics cancarried out as described in “A unified view of polymer, dumbbell, andoligonucleotide DNA nearest-neighbor thermodynamics” John SantaLucia,Jr., PNAS Feb. 17, 1998 vol. 95 no. 4 1460-1465 (which is incorporatedby reference herein for this description).

The term “oligonucleotide” is used to refer to a nucleic acid that isrelatively short, generally shorter than 200 nucleotides, moreparticularly, shorter than 100 nucleotides, most particularly, shorterthan 50 nucleotides. Typically, oligonucleotides are single-stranded DNAmolecules.

The term “primer” refers to an oligonucleotide that is capable ofhybridizing (also termed “annealing”) with a nucleic acid and serving asan initiation site for nucleotide (RNA or DNA) polymerization underappropriate conditions (i.e., in the presence of four differentnucleoside triphosphates and an agent for polymerization, such as DNA orRNA polymerase or reverse transcriptase) in an appropriate buffer and ata suitable temperature. The appropriate length of a primer depends onthe intended use of the primer, but primers are typically at least 7nucleotides long and, in some embodiments, range from 10 to 30nucleotides, or, in some embodiments, from 10 to 60 nucleotides, inlength. In some embodiments, primers can be, e.g., 15 to 50 nucleotideslong. Short primer molecules generally require cooler temperatures toform sufficiently stable hybrid complexes with the template. A primerneed not reflect the exact sequence of the template but must besufficiently complementary to hybridize with a template.

A primer is said to anneal to another nucleic acid if the primer, or aportion thereof, hybridizes to a nucleotide sequence within the nucleicacid. The statement that a primer hybridizes to a particular nucleotidesequence is not intended to imply that the primer hybridizes eithercompletely or exclusively to that nucleotide sequence. For example, insome embodiments, amplification primers used herein are said to “annealto” or be “specific for” a nucleotide sequence.” This descriptionencompasses primers that anneal wholly to the nucleotide sequence, aswell as primers that anneal partially to the nucleotide sequence.

The term “primer pair” refers to a set of primers including a 5′“upstream primer” or “forward primer” that hybridizes with thecomplement of the 5′ end of the DNA sequence to be amplified and a 3′“downstream primer” or “reverse primer” that hybridizes with the 3′ endof the sequence to be amplified. As will be recognized by those of skillin the art, the terms “upstream” and “downstream” or “forward” and“reverse” are not intended to be limiting, but rather provideillustrative orientations in some embodiments.

A “probe” is a nucleic acid capable of binding to a target nucleic acidof complementary sequence through one or more types of chemical bonds,generally through complementary base pairing, usually through hydrogenbond formation, thus forming a duplex structure. The probe can belabeled with a detectable label to permit facile detection of the probe,particularly once the probe has hybridized to its complementary target.Alternatively, however, the probe may be unlabeled, but may bedetectable by specific binding with a ligand that is labeled, eitherdirectly or indirectly. Probes can vary significantly in size.Generally, probes are at least 7 to 15 nucleotides in length. Otherprobes are at least 20, 30, or 40 nucleotides long. Still other probesare somewhat longer, being at least 50, 60, 70, 80, or 90 nucleotideslong. Yet other probes are longer still, and are at least 100, 150, 200or more nucleotides long. Probes can also be of any length that iswithin any range bounded by any of the above values (e.g., 15-20nucleotides in length).

The primer or probe can be perfectly complementary to the targetnucleotide sequence or can be less than perfectly complementary. In someembodiments, the primer has at least 65% identity to the complement ofthe target nucleotide sequence over a sequence of at least 7nucleotides, more typically over a sequence in the range of 10-30nucleotides, and, in some embodiments, over a sequence of at least 14-25nucleotides, and, in some embodiments, has at least 75% identity, atleast 85% identity, at least 90% identity, or at least 95%, 96%, 97%,98%, or 99% identity. It will be understood that certain bases (e.g.,the 3′ base of a primer) are generally desirably perfectly complementaryto corresponding bases of the target nucleotide sequence. Primer andprobes typically anneal to the target sequence under stringenthybridization conditions.

As used herein with reference to a portion of a primer or a nucleotidesequence within the primer, the term “specific for” a nucleic acid,refers to a primer or nucleotide sequence that can specifically annealto the target nucleic acid under suitable annealing conditions.

Amplification according to the present teachings encompasses any meansby which at least a part of at least one target nucleic acid isreproduced, typically in a template-dependent manner, including withoutlimitation, a broad range of techniques for amplifying nucleic acidsequences, either linearly or exponentially.

Illustrative means for performing an amplifying step include PCR,nucleic acid strand-based amplification (NASBA), two-step multiplexedamplifications, rolling circle amplification (RCA), and the like,including multiplex versions and combinations thereof, for example butnot limited to, OLA/PCR, PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR,LCR/PCR, PCR/LCR (also known as combined chain reaction—CCR),helicase-dependent amplification (HDA), and the like. Descriptions ofsuch techniques can be found in, among other sources, Ausbel et al.; PCRPrimer: A Laboratory Manual, Diffenbach, Ed., Cold Spring Harbor Press(1995); The Electronic Protocol Book, Chang Bioscience (2002); Msuih etal., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid ProtocolsHandbook, R. Rapley, ed., Humana Press, Totowa, N.J. (2002); Abramson etal., Curr Opin Biotechnol. 1993 February; 4(1):41-7, U.S. Pat. No.6,027,998; U.S. Pat. No. 6,605,451, Barany et al., PCT Publication No.WO 97/31256; Wenz et al., PCT Publication No. WO 01/92579; Day et al.,Genomics, 29(1): 152-162 (1995), Ehrlich et al., Science 252:1643-50(1991); Innis et al., PCR Protocols: A Guide to Methods andApplications, Academic Press (1990); Favis et al., Nature Biotechnology18:561-64 (2000); and Rabenau et al., Infection 28:97-102 (2000);Belgrader, Barany, and Lubin, Development of a Multiplex LigationDetection Reaction DNA Typing Assay, Sixth International Symposium onHuman Identification, 1995 (available on the world wide web at:promega.com/geneticidproc/ussymp6proc/blegrad.html-); LCR KitInstruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002;Barany, Proc. Natl. Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook,Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res.27:e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66(2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl.Acid Res. 20:1691-96 (1992); Polstra et al., BMC Inf. Dis. 2:18- (2002);Lage et al., Genome Res. 2003 February; 13(2):294-307, and Landegren etal., Science 241:1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002November; 2(6):542-8., Cook et al., J Microbiol Methods. 2003 May;53(2):165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 February;12(1):21-7, U.S. Pat. Nos. 5,830,711, 6,027,889, 5,686,243, PCTPublication No. WO0056927A3, and PCT Publication No. WO9803673A1.

In some embodiments, amplification comprises at least one cycle of thesequential procedures of: annealing at least one primer withcomplementary or substantially complementary sequences in at least onetarget nucleic acid; synthesizing at least one strand of nucleotides ina template-dependent manner using a polymerase; and denaturing thenewly-formed nucleic acid duplex to separate the strands. The cycle mayor may not be repeated. Amplification can comprise thermocycling or canbe performed isothermally.

“Nested amplification” refers the use of more than two primers toamplify a target nucleic acid.

“Hemi-nested amplification” refers to the use of more than one primer(e.g., two or three) that anneal at one end of a target nucleotidesequence.

“Fully nested amplification” refers to the use of more than one primerthat anneal at each end of a target nucleotide sequence.

With reference to nested amplification, the multiple primers that annealat one end of an amplicon are differentiated by using the terms “inner,”“outer,” and “intermediate.”

An “outer primer” refers to a primer that that anneals to a sequencecloser to the end of the target nucleotide sequence than another primerthat anneals at that same end of the target nucleotide sequence. In someembodiments, the outer primer sequence defines the end of the ampliconproduced from the target nucleic acid. The “outer primer” is alsoreferred to herein as a “flanking primer.”

An “inner primer” refers to a primer that that anneals to a sequencecloser to the middle of the target nucleotide sequence than anotherprimer that anneals at that same end of the target nucleotide sequence.

The term “intermediate primer” is used herein with reference to nestamplification in which at least three primers that anneal at one end ofa target nucleotide sequence are used. An intermediate primer is onethat anneals to a sequence in between an inner primer and an outerprimer.

As used herein, the term “adjacent to” is used to refer to sequencesthat are in sufficiently close proximity for the methods to work. Insome embodiments, sequences that are adjacent to one another areimmediately adjacent, with no intervening nucleotides.

A “multiplex amplification reaction” is one in which two or more nucleicacids distinguishable by sequence are amplified simultaneously.

The term “qPCR” is used herein to refer to quantitative real-timepolymerase chain reaction (PCR), which is also known as “real-time PCR”or “kinetic polymerase chain reaction;” all terms refer to PCR withreal-time signal detection.

A “reagent” refers broadly to any agent used in a reaction, other thanthe analyte (e.g., nucleic acid being analyzed). Illustrative reagentsfor a nucleic acid amplification reaction include, but are not limitedto, buffer, metal ions, polymerase, reverse transcriptase, primers,template nucleic acid, nucleotides, labels, dyes, nucleases, dNTPs, andthe like. Reagents for enzyme reactions include, for example,substrates, cofactors, buffer, metal ions, inhibitors, and activators.

The term “label,” as used herein, refers to any atom or molecule thatcan be used to provide a detectable and/or quantifiable signal. Inparticular, the label can be attached, directly or indirectly, to anucleic acid or protein. Suitable labels that can be attached to probesinclude, but are not limited to, radioisotopes, fluorophores,chromophores, mass labels, electron dense particles, magnetic particles,spin labels, molecules that emit chemiluminescence, electrochemicallyactive molecules, enzymes, cofactors, and enzyme substrates.

The term “dye,” as used herein, generally refers to any organic orinorganic molecule that absorbs electromagnetic radiation.

The term “fluorescent dye,” as used herein, generally refers to any dyethat emits electromagnetic radiation of longer wavelength by afluorescent mechanism upon irradiation by a source of electromagneticradiation, such as a lamp, a photodiode, or a laser or anotherfluorescent dye.

General Approach for Increasing Amplification Efficiency

U.S. Pat. No. 8,252,558 and Harris et al., BioTechniques 54:93-97(February 2013) teach a form of nested PCR, termed “Polymerase ChainDisplacement Reaction” (PCDR) (both documents are incorporated byreference herein for this description). In PCDR, when extension occursfrom an outer primer, it displaces the extension strand produced from aninner primer because the reaction employs a polymerase that has stranddisplacement activity. In theory, this allows a greater than 2-foldincrease of amplification product for each amplification cycle andtherefore increased sensitivity and speed over conventional PCR. Inpractice, every amplicon created from a nested primer no longer containsa primer annealing site for the outer primer. Accordingly, PCDR cannotsustain a greater than 2-fold increase of amplification product for eachamplification cycle for very many cycles. For this reason, PCDR offersonly modest reduction in the number of amplification cycles (e.g., fromabout 23 to about 20) needed to detect a target nucleic acid. Bycontrast, Table 1 below shows that a sustained quadrupling per cycle(4^(number of cycles)) should halve the number of cycles needed to havethe same amplification as a doubling per cycle. A sustained 6-foldreplication per cycle should achieve in 15 cycles what would take 40normal PCR cycles.

TABLE 1 Degree of Amplification With Different “Bases” base cycle 2 3 45 6 0 1 1 1 1 1 1 2 3 4 5 6 2 4 9 16 25 36 3 8 27 64 125 216 4 16 81 256625 1296 5 32 243 1024 3125 7776 6 64 729 4096 15625 46656 7 128 218716384 78125 279936 8 256 6561 65536 390625 1679616 9 512 19683 2621441953125 10077696 10 1024 59049 1048576 9765625 60466176 11 2048 1771474194304 48828125 3.63E+08 12 4096 531441 16777216 2.44E+08 2.18E+08 138192 1594323 67108864 1.22E+08 1.31E+10 14 16384 4782969 2.68E+08 6.1E+08 7.84E+10 15 32768 14348907 1.07E+08 3.05E+10  4.7E+11 16 6553643046721 4.28E+08 1.43E+11 2.82E+12 17 131072 1.26E+08 1.72E+10 7.63E+111.68E+13 18 262144 3.67E+08 6.67E+10 3.61E+12 1.02E+14 19 5242881.16E+08 2.74E+11 1.61E+13 6.08E+14 20 1048576 3.46E+08  1.1E+129.54E+13 3.66E+15 21 2097152 1.06E+10  4.4E+12 4.77E+14 2.18E+15 224194304 3.14E+10 1.76E+13 2.38E+15 1.32E+17 23 8388608 9.41E+10 7.04E+131.18E+16  7.8E+17 24 16777216 2.82E+11 2.81E+14 5.84E+16 4.74E+18 2533554432 8.47E+11 1.13E+15 2.88E+17 2.64E+19 26 67108864 2.64E+12 4.6E+15 1.48E+18 1.71E+20 27 1.34E+08 7.83E+12  1.6E+16 7.45E+181.02E+21 28 2.68E+08 2.28E+13 7.21E+16 3.73E+18 8.14E+21 29 4.37E+088.86E+13 2.88E+17 1.84E+20 3.68E+22 30 1.07E+08 2.06E+14 1.14E+186.31E+20 2.21E+23 31 2.14E+08 4.18E+14 4.81E+18 4.66E+21 1.33E+24 324.28E+08 1.85E+15 1.84E+19 2.33E+22 7.86E+24 33 8.48E+08 4.44E+167.38E+19 1.16E+23 4.78E+25 34 1.72E+10 1.67E+16 2.85E+20 4.82E+232.87E+25 35 3.44E+10   5E+16 1.18E+21 2.91E+24 1.72E+27 36 6.87E+10 1.5E+17 4.72E+21 1.46E+25 1.03E+28 37 1.37E+11  4.5E+17 1.88E+227.29E+25 6.18E+28 38 2.76E+11 1.35E+18 7.44E+22 3.54E+26 3.71E+29 39 6.6E+11 4.05E+18 3.02E+23 1.82E+27 2.23E+30 40  1.1E+12 1.22E+191.21E+24 8.09E+27 1.34E+31

A key to sustaining a greater than 2-fold increase of amplificationproduct for each amplification cycle is to design the inner (nested)primer so that the extension product of the inner (nested) primercontains the outer (flanking) primer sequence. FIG. 1 shows a scheme inwhich fully nested PCR is carried out using a forward inner and outerprimer and a reverse inner and outer primer. The “flap” formed wheninner primer anneals to template contains the outer primer sequence sothat each of the four new strands generated from the two templatestrands extends from (and includes) either the forward outer primersequence (or its complement) through (and including) the reverse outerprimer sequence. However, more is required than simply appending theouter primer sequence to the 5′ end of the inner primer because, whenthe inner primer anneals, the appended sequence would immediately alsoanneal and block the outer primer from annealing. A solution to thisproblem is to use an additional 5′ add-on to the inner primer (i.e., asequence in addition to the outer primer sequence) together with anoligonucleotide complementary to both sequences, which is referred toherein as a “clamp.” For ease of discussion, the add-on sequence istermed a “clamp sequence.” This configuration is shown in FIG. 2.

The clamp sequence, c, is not homologous to the template (d′ region).Here c-a/c′-a′ is more stable than a-b/a′-b′. In some embodiments, thiscan be achieved by employing a sequence c that is long relative to a,GC-rich (i.e., more GC-rich than a), or contains one or more stabilizingbases, when a does not contain such bases, or more stabilizing basesthan in a. In some embodiments, this can be achieved by employing asequence c that is long relative to b, GC-rich (i.e., more GC-rich thanb), or contains one or more stabilizing bases, when b does not containsuch bases, or more stabilizing bases than in b. In some embodiments, astabilizing base can be included in the a region of c-a-b, as well as inthe a′ region of c′-a′ to enhance the stability of c-a/c′-a′, relativeto a-b/a′-b′. Alternatively or in addition, b can contain one or moredestabilizing bases, such as inosine. The outer primer remains sequencea. In this case, sequence a′ in the template remains available for theouter primer. The c′-a′ clamp and the 5′ end of the inner primer willrapidly anneal at a higher temperature than any of the other sequences,and therefore it is not necessary that the c′-a′ clamp be linked to theinner primer so as to form a hairpin structure. However, in someembodiments, the use of an inner primer having this type of hairpinstructure may increase the speed of the reaction.

In some embodiments, the c sequence in the inner primer is preferablynot to be copied during PCR. If it is, then these new templates willhave a c′-a′ tail that will create with the inner primer ac-a-b/c′-a′-b′ duplex that would “win-out” in the strand displacementcontest over the other possible structures and, again, prevent theflanking primer, sequence a, from annealing. To prevent this copying,the c sequence can be made from RNA (or 2′-O-methyl RNA, which isrelatively easy to make synthetically), which DNA polymerase cannot copywell. This c sequence can be made from any bases capable ofbase-pairing, but not capable of being copied.

In some embodiments, the clamp oligonucleotide (a′-c′) is blocked toextension at the 3′ end, e.g., by virtue of lacking a 3′ hydroxyl groupor using a chemical blocking moiety, which can improve the specificityof the amplification.

Samples

Nucleic acid-containing samples can be obtained from biological sourcesand prepared using conventional methods known in the art. In particular,nucleic useful in the methods described herein can be obtained from anysource, including unicellular organisms and higher organisms such asplants or non-human animals, e.g., canines, felines, equines, primates,and other non-human mammals, as well as humans. In some embodiments,samples may be obtained from an individual suspected of being, or knownto be, infected with a pathogen, an individual suspected of having, orknown to have, a disease, such as cancer, or a pregnant individual.

Nucleic acids can be obtained from cells, bodily fluids (e.g., blood, ablood fraction, urine, etc.), or tissue samples by any of a variety ofstandard techniques. In some embodiments, the method employs samples ofplasma, serum, spinal fluid, lymph fluid, peritoneal fluid, pleuralfluid, oral fluid, and external sections of the skin; samples from therespiratory, intestinal genital, or urinary tracts; samples of tears,saliva, blood cells, stem cells, or tumors. Samples can be obtained fromlive or dead organisms or from in vitro cultures. Illustrative samplescan include single cells, paraffin-embedded tissue samples, and needlebiopsies. In some embodiments, the nucleic acids analyzed are obtainedfrom a single cell.

Nucleic acids of interest can be isolated using methods well known inthe art. The sample nucleic acids need not be in pure form, but aretypically sufficiently pure to allow the steps of the methods describedherein to be performed.

Target Nucleic Acids

Any target nucleic acid that can detected by nucleic acid amplificationcan be detected using the methods described herein. In typicalembodiments, at least some nucleotide sequence information will be knownfor the target nucleic acids. For example, if the amplification reactionemployed is PCR, sufficient sequence information is generally availablefor each end of a given target nucleic acid to permit design of suitableamplification primers.

The targets can include, for example, nucleic acids associated withpathogens, such as viruses, bacteria, protozoa, or fungi; RNAs, e.g.,those for which over- or under-expression is indicative of disease,those that are expressed in a tissue- or developmental-specific manner;or those that are induced by particular stimuli; genomic DNA, which canbe analyzed for specific polymorphisms (such as SNPs), alleles, orhaplotypes, e.g., in genotyping. Of particular interest are genomic DNAsthat are altered (e.g., amplified, deleted, and/or mutated) in geneticdiseases or other pathologies; sequences that are associated withdesirable or undesirable traits; and/or sequences that uniquely identifyan individual (e.g., in forensic or paternity determinations).

Primer Design

Primers suitable for nucleic acid amplification are sufficiently long toprime the synthesis of extension products in the presence of a suitablenucleic acid polymerase. The exact length and composition of the primerwill depend on many factors, including, for example, temperature of theannealing reaction, source and composition of the primer, and where aprobe is employed, proximity of the probe annealing site to the primerannealing site and ratio of primer:probe concentration. For example,depending on the complexity of the target nucleic acid sequence, anoligonucleotide primer typically contains in the range of about 10 toabout 60 nucleotides, although it may contain more or fewer nucleotides.The primers should be sufficiently complementary to selectively annealto their respective strands and form stable duplexes.

In general, one skilled in the art knows how to design suitable primerscapable of amplifying a target nucleic acid of interest. For example,PCR primers can be designed by using any commercially available softwareor open source software, such as Primer3 (see, e.g., Rozen and Skaletsky(2000) Meth. Mol. Biol., 132: 365-386; www.broad.mit.edu/node/1060, andthe like) or by accessing the Roche UPL website. The amplicon sequencesare input into the Primer3 program with the UPL probe sequences inbrackets to ensure that the Primer3 program will design primers oneither side of the bracketed probe sequence.

Primers may be prepared by any suitable method, including, for example,direct chemical synthesis by methods such as the phosphotriester methodof Narang et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiestermethod of Brown et al. (1979) Meth. Enzymol. 68: 109-151; thediethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett.,22: 1859-1862; the solid support method of U.S. Pat. No. 4,458,066 andthe like, or can be provided from a commercial source. Primers may bepurified by using a Sephadex column (Amersham Biosciences, Inc.,Piscataway, N.J.) or other methods known to those skilled in the art.Primer purification may improve the sensitivity of the methodsdescribedherein.

Outer Primer

FIG. 2 shows how a two-primer set anneals to a first template strand atone end of a target nucleotide sequence. For ease of discussion, thisprimer set can be considered to be a “forward” primer set. The outerprimer includes a sequence a that specifically hybridizes to firsttemplate strand sequence a′. FIG. 3 shows how a two-primer set annealsto a second template strand at the opposite end of the target nucleotidesequence. For ease of discussion, this primer set can be considered tobe a “reverse” primer set. Here, the outer primer includes a sequence ethat specifically hybridizes to first template strand sequence e′. FIGS.4 and 5 show illustrative “forward” and “reverse” three-primer sets. InFIG. 4, the forward outer primer includes a sequence d that specificallyhybridizes to first template strand sequence d′. In FIG. 5, the forwardouter primer includes a sequence d that specifically hybridizes to firsttemplate strand sequence d′. In general, the considerations fordesigning suitable outer primers do not differ from those for designingouter primers for use in conventional nested PCR. Notably, in someembodiments, the T_(m) of any primer sequence that is “outer” relativeto another primer sequence (e.g., an inner or intermediate primersequence) is preferably lower than the T_(m) of the inner (orintermediate) primer sequence. Thus, for example, during the downtemperature ramp of PCR, the inner primer can anneal and begin extensionbefore the outer primer; otherwise premature extension of the outerprimer would block the target site of the inner primer and prevent itsannealing. More specifically, in the embodiment shown in FIG. 2, primersequence a would have a T_(m) less than that of primer sequence b.Similarly, in the embodiment shown in FIG. 3, primer sequence e wouldhave a lower T_(m) than primer sequence f. In some embodiments, theT_(m) differences are at least about 4 degrees, generally in the rangeof about 4 to about 20 degrees C. In some embodiments, the T_(m)differences are in the range of about 4 to about 15 degrees C. However,the T_(m) of the outer primer is generally high enough to maintainefficient PCR, e.g., in some embodiments, the T_(m) of the outer primeris at least 40 degrees C. T_(m) can be adjusted by adjusting the lengthof a sequence, the G-C content, and/or by including stabilizing ordestabilizing base(s) in the sequence.

“Stabilizing bases” include, e.g., stretches of peptide nucleic acids(PNAs) that can be incorporated into DNA oligonucleotides to increaseduplex stability. Locked nucleic acids (LNAs) and unlocked nucleic acids(UNAs) are analogues of RNA that can be easily incorporated into DNAoligonucleotides during solid-phase oligonucleotide synthesis, andrespectively increase and decrease duplex stability. Suitablestabilizing bases also include modified DNA bases that increase thestability of base pairs (and therefore the duplex as a whole). Thesemodified bases can be incorporated into oligonucleotides duringsolid-phase synthesis and offer a more predictable method of increasingDNA duplex stability. Examples include AP-dC (G-clamp) and2-aminoadenine, as well as 5-methylcytosine and C(5)-propynylcytosine(replacing cytosine), and C(5)-propynyluracil (replacing thymine).

“Destabilizing bases” are those that destabilize double-stranded DNA byvirtue of forming less stable base pairs than the typical A-T and/or G-Cbase pairs. Inosine (I) is a destabilizing base because it pairs withcytosine (C), but an I-C base pair is less stable than a G-C base pair.This lower stability results from the fact that inosine is a purine thatcan make only two hydrogen bonds, compared to the three hydrogen bondsof a G-C base pair. Other destabilizing bases are known to, or readilyidentified by, those of skill in the art.

Inner Primer of a Two-Primer Set

Referring to FIG. 2, the inner primer in a forward two-primer setincludes a single-stranded primer sequence b that specificallyhybridizes to first template strand sequence b′, wherein b′ is adjacentto, and 5′ of, a′, and wherein single-stranded primer sequence b islinked at its 5′ end to a first strand of a double-stranded primersequence. This first stand includes: a primer sequence a adjacent to,and 5′ of, single-stranded primer sequence b; and a clamp sequence cadjacent to, and 5′ of, primer sequence a, wherein clamp sequence c isnot complementary to a first strand template sequence d′, which isadjacent to, and 3′ of, first strand template sequence a′. In someembodiments, the T_(m) of combined sequence c-a (the hyphen is used inthis context to denote the combined nucleic acid sequence made up ofsequences c and a) in double-stranded form (i.e., c-a/c′-a′), is greaterthan that of combined sequence a-b, in double stranded form (i.e.,a-b/a′-b′). This is readily achieved, e.g., by making combined sequencec-a longer and/or more GC-rich than combined sequence a-b, and/ordesigning combined sequence c-a to include more stabilizing bases thancombined sequence a-b (the requirement for “more” includes the situationin which sequence a-b contains no G-C basepairs and/or no stabilizingbases). Alternatively or in addition, combined sequence a-b can bedesigned to include more destabilizing bases than combined sequence c-a(the requirement for “more” includes the situation in which sequence c-acontains no destabilizing bases). In some embodiments, a′-c′ is blockedto extension at its 3′ end.

The forward two-primer set can be employed with a simple conventionalreverse primer for a hemi-nested amplification or with a reversetwo-primer set.

Referring to FIG. 3, the inner primer in a reverse two-primer setincludes a single-stranded primer sequence f that specificallyhybridizes to first template strand sequence f′, wherein f′ is adjacentto, and 5′ of, e′, and wherein single-stranded primer sequence f islinked at its 5′ end to a first strand of a double-stranded primersequence. This first stand includes: a primer sequence e adjacent to,and 5′ of, single-stranded primer sequence f; and a clamp sequence gadjacent to, and 5′ of, primer sequence e, wherein clamp sequence g isnot complementary to a first strand template sequence h′, which isadjacent to, and 3′ of, first strand template sequence e′. In someembodiments, the T_(m) of combined sequence g-e, in double-stranded form(i.e., g-e/g′-e′) is greater than that of combined sequence e-f, indouble-stranded form (i.e., e-f/e′-f) This is readily achieved, e.g., bymaking combined sequence g-e longer and/or more GC-rich than combinedsequence e-f, and/or designing combined sequence the T_(m) of combinedsequence g-e, in double-stranded form is greater than that of combinedsequence e-f, in double-stranded form to include more stabilizing basesthan combined sequence e-f (the requirement for “more” includes thesituation in which sequence e-f contains no G-C base pairs and/or nostabilizing bases). Alternatively or in addition, combined sequence e-fcan be designed to include more destabilizing bases than combinedsequence g-e (the requirement for “more” includes the situation in whichsequence g-e contains no destabilizing bases). In some embodiments,e′-g′ is blocked to extension at its 3′ end.

In some embodiments, clamp sequence(s) c and g, if present, is/are notcapable of being copied during amplification. RNA or an RNA analog,e.g., a hydrolysis-resistant RNA analog, can be employed to provide therequired base pairing to form the double-stranded clamp sequence withoutbeing copied by a DNA-dependent polymerase during amplification. Themost common RNA analogues is 2′-O-methyl-substituted RNA. Other nucleicacid analogues that can base pair specifically but cannot be copiedinclude locked nucleic acid (LNA) or BNA (Bridged Nucleic Acid),morpholino, and peptide nucleic acid (PNA). Although theseoligonucleotides have a different backbone sugar or, in the case of PNA,an amino acid residue in place of the ribose phosphate, they still bindto RNA or DNA according to Watson and Crick pairing, but are immune tonuclease activity. They cannot be synthesized enzymatically and can onlybe obtained synthetically using phosphoramidite strategy or, for PNA,methods of peptide synthesis.

If desired, the clamp sequence c can be covalently linked tocomplementary sequence c′ so that a-c/a-c′ is formed from a hairpinstructure; however, this is not necessary for efficient formation of thedouble-stranded clamp portion of the primer. Similarly, the clampsequence g can, but need not, be covalently linked to complementarysequence g′ so that e-g/e′-g′ is formed from a hairpin structure.

Primers of a Three-Primer Set

In some embodiments, a third primer may be employed at one or both endsof a target nucleic acid sequence to further increase the number ofcopies produced in each cycle of amplification. FIGS. 4 and 5 showillustrative “forward” and “reverse” three-primer sets. A three-primerset includes an outer primer as discussed above and an intermediateprimer that is essentially the same in structure as the inner primerdiscussed above. The additional primer is an inner primer which isdesigned to hybridize to the template strand 5′ of the intermediateprimer.

The inner primer in a forward three-primer set includes asingle-stranded primer sequence b that specifically hybridizes to firsttemplate strand sequence b′, wherein b′ is adjacent to, and 5′ of, a′.Single-stranded primer sequence b is linked at its 5′ end to a firststrand of a double-stranded primer sequence comprising: a primersequence a adjacent to, and 5′ of, single-stranded primer sequence b, aprimer sequence d adjacent to, and 5′ of, primer sequence a, and a clampsequence c2 adjacent to, and 5′ of, primer sequence d, wherein clampsequence c2 is not complementary to first strand template sequence i′.Clamp sequence c2 can be the same as, or different from, the clampsequence used in the inner primer (c1). In preferred embodiments, c1 andc2 are different sequences. Similar considerations apply to the designof the inner primer in a three-primer set as discussed above withrespect to the inner primer in a two primer set, and the inner primer ina reverse three-primer set (shown in FIG. 5) has the same structure asthe inner primer in a forward three-primer set. One or more (or all) ofthe clamp oligonucleotides (d′-c1′ and a′-d′-c2′ in FIG. 4 and h′-g1′and e′-h′-g2′ in FIG. 5) can be blocked to extension at their 3′ ends.The forward three-primer set can be employed with a simple conventionalreverse primer for a hemi-nested amplification, with a reversetwo-primer set, or with a reverse three-primer set.

In some embodiments, the order of primer annealing and extension iscontrolled based on the T_(m) of the primer sequences so that any primerthat is “inner” with respect to another primer anneals and beginsextension before that other primer. Thus, for example, in a two-primerset, the inner primer anneals and begins extension before the outerprimer, and in a three-primer set, the inner primer anneals and beginsextension before the intermediate primer, and the intermediate primeranneals and begins extension before the outer primer. For example, inthe embodiment shown in FIG. 4, the T_(m)'s of the primer sequenceswould have the relationship: T_(m) of d<T_(m) of a<T_(m) of b. In theembodiment shown in FIG. 5, the T_(m)'s of the primer sequences wouldhave the relationship: T_(m) of h<T_(m) of e<T_(m) of f. As noted above,T_(m)'s are a function of sequence length, C-G content, and the,optional, presence of stabilizing and/or destabilizing bases.

Further nested primers can be designed based on the principles discussedabove.

Polymerase

The disclosed methods make the use of a polymerase for amplification. Insome embodiments, the polymerase is a DNA polymerase that lacks a 5′ to3′ exonuclease activity. The polymerase is used under conditions suchthat the strand extending from a first primer can be displaced bypolymerization of the forming strand extending from a second primer thatis “outer” with respect to the first primer. Conveniently, thepolymerase is capable of displacing the strand complementary to thetemplate strand, a property termed “strand displacement.” Stranddisplacement results in synthesis of multiple copies of the targetsequence per template molecule. In some embodiments, the DNA polymerasefor use in the disclosed methods is highly processive. Exemplary DNApolymerases include variants of Taq DNA polymerase that lack 5′ to 3′exonuclease activity, e.g., the Stoffel fragment of Taq DNA polymerase(ABI), SD polymerase (Bioron), mutant Taq lacking 5′ to 3′ exonucleaseactivity described in U.S. Pat. No. 5,474,920, Bca polymerase (Takara),Pfx50 polymerase (Invitrogen), Tfu DNA polymerase (Qbiogene). Ifthermocycling is to be carried out (as in PCR), the DNA polymerase ispreferably a thermostable DNA polymerase. Table 2 below listspolymerases available from New England Biolabs that have no 5′ to 3′exonuclease activity, but that have strand displacement activityaccompanied by thermal stability.

TABLE 2 Thermostable Stand-Displacing Polymerases Lacking 5′ to 3′Exonuclease Activity 5′->3′ Strand Thermal Polymerase ExonucleaseDisplacement Stability Bst DNA Polymerase, − ++++ + Large Fragment BsuDNA Polymerase, − ++ − Large Fragment DEEP VENT_(R) ™ − ++ ++++ DNAPolymerase DEEP VENT_(R) ™ (exo-) − +++ ++++ DNA Polymerase KlenowFragment (3′→5′ exo-) − +++ − DNA Polymerase I, − ++ − Large (Klenow)Fragment M-MuLV Reverse − +++ − Transcriptase phi29 DNA Polymerase −+++++ − THERMINATOR ™ DNA − + ++++ Polymerase VENT_(R) ® DNA − ++^(θ)+++ Polymerase VENT_(R) ® (exo-) − +++^(θ) +++ DNA PolymeraseIn some embodiments, the DNA polymerase comprises a fusion between Taqpolymerase and a portion of a topoisomerase, e.g., TOPOTAQ™ (FidelitySystems, Inc.).

Strand displacement can also be facilitated through the use of a stranddisplacement factor, such as a helicase. Any DNA polymerase that canperform strand displacement in the presence of a strand displacementfactor is suitable for use in the disclosed method, even if the DNApolymerase does not perform strand displacement in the absence of such afactor. Strand displacement factors useful in the methods describedherein include BMRF1 polymerase accessory subunit (Tsurumi et al., J.Virology 67(12):7648-7653 (1993)), adenovirus DNA-binding protein(Zijderveld and van der Vliet, J. Virology 68(2):1158-1164 (1994)),herpes simplex viral protein ICP8 (Boehmer and Lehman, J. Virology67(2):711-715 (1993); Skaliter and Lehman, Proc. Natl. Acad. Sci. USA91(22):10665-10669 (1994)), single-stranded DNA binding proteins (SSB;Rigler and Romano, J. Biol. Chem. 270:8910-8919 (1995)), and calf thymushelicase (Siegel et al., J. Biol. Chem. 267:13629-13635 (1992)).Helicase and SSB are available in thermostable forms and thereforesuitable for use in PCR.

Amplification

The primer sets described above are contacted with sample nucleic acidsunder conditions wherein the primers anneal to their template strands,if present. The desired nucleic acid amplification method is carried outusing a DNA polymerase lacking 5′-3′ exonuclease activity that iscapable of strand displacement under the reaction conditions employed.This amplification produces amplicons that include the sequences of allprimers employed in the amplification reaction. The primer sets canconveniently be added to the amplification mixture in the form ofseparate oligonucleotides. For example, the two-primer set can consistof three oligonucleotides (assuming that the inner primer does notinclude a hairpin structure) and the three-primer set can consist offive oligonucleotides (assuming that neither the inner, nor theintermediate, primers include a hairpin structure).

For hemi-nested amplification using a two-primer set, as described abovea rate of up to 3^(number of cycles) during the exponential phase of PCRcan be achieved. Amplification using a hemi-nested two-primer set canreduce the number of amplification cycles required to detect asingle-copy nucleic acid by about 12% to about 42% (e.g., by 37%). Thisfacilitates detection of a single copy nucleic acid in a biologicalsample within about 23-27 amplification cycles (which might otherwiserequire 40 or more cycles). In some embodiments, hemi-nested, two-primerset PCR facilitates detection of a single copy nucleic acid in abiological sample in 23, 24, 25, 26, or 27 amplification cycles.

Table 3 below shows the number of cycles needed to amplify a single-copynucleic acid to 10¹² copies using the different embodiment describedherein. For fully-nested amplification using a two-primer set, asdescribed above, a rate of up to 6^(number of cycles) during theexponential phase of PCR can be achieved. Amplification using afully-nested two-primer set can reduce the number of amplificationcycles required to detect a single-copy nucleic acid by about 36% toabout 66% (e.g., by 61%). This facilitates detection of a single copynucleic acid in a biological sample within about 13-17 amplificationcycles. In some embodiments, fully-nested, two-primer set PCRfacilitates detection of a single copy nucleic acid in a biologicalsample in 13, 14, 15, 16, or 17 amplification cycles.

TABLE 3 Reduction in Number of Cycles Needed for Amplification as aFunction of PCR Base number of cycles needed % eduction upper boundlower bound to reach of cycles reduction reduction PCR base 10∧12 copiesneeded (+5%) (−25%) 2 39.86 na na na 3 25.15 37% 42% 12% 4 19.93 50% 55%25% 6 15.42 61% 66% 36% 8 13.29 67% 72% 42%

For hemi-nested amplification using a three-primer set, as describedabove, a rate of up to 4^(number of cycles) during the exponentialphaseof PCR can be achieved. Amplification using a hemi-nested three-primerset can reduce the number of amplification cycles required to detect asingle-copy nucleic acid by about 25% to about 55% (e.g., by 50%). Thisfacilitates detection of a single copy nucleic acid in a biologicalsample within about 20 amplification cycles (which might otherwiserequire 40 or more cycles). In some embodiments, hemi-nested,three-primer set PCR facilitates detection of a single copy nucleic acidin a biological sample in 18, 19, 20, 21, or 22 amplification cycles.

For fully-nested amplification using a three-primer set, as describedabove, a rate of up to 8^(number of cycles) during the exponential phaseof PCR can be achieved. Amplification using a fully-nested three-primerset can reduce the number of amplification cycles required to detect asingle-copy nucleic acid by about 42% to about 72% (e.g., by 67%). Thisfacilitates detection of a single copy nucleic acid in a biologicalsample within about 11-15 amplification cycles. In some embodiments,fully-nested, three-primer set PCR facilitates detection of a singlecopy nucleic acid in a biological sample in 9, 10, 11, 12, or 13amplification cycles.

In some embodiments, the amplification step is performed using PCR. Forrunning real-time PCR reactions, reaction mixtures generally contain anappropriate buffer, a source of magnesium ions (Mg²⁺) in the range ofabout 1 to about 10 mM, e.g., in the range of about 2 to about 8 mM,nucleotides, and optionally, detergents, and stabilizers. An example ofone suitable buffer is TRIS buffer at a concentration of about 5 mM toabout 85 mM, with a concentration of 10 mM to 30 mM preferred. In oneembodiment, the TRIS buffer concentration is 20 mM in the reaction mixdouble-strength (2×) form. The reaction mix can have a pH range of fromabout 7.5 to about 9.0, with a pH range of about 8.0 to about 8.5 astypical. Concentration of nucleotides can be in the range of about 25 mMto about 1000 mM, typically in the range of about 100 mM to about 800mM. Examples of dNTP concentrations are 100, 200, 300, 400, 500, 600,700, and 800 mM. Detergents such as Tween 20, Triton X 100, and NonidetP40 may also be included in the reaction mixture. Stabilizing agentssuch as dithiothreitol (DTT, Cleland's reagent) or mercaptoethanol mayalso be included. In addition, master mixes may optionally contain dUTPas well as uracil DNA glycosylase (uracil-N-glycosylase, UNG). A mastermix is commercially available from Applied Biosystems, Foster City,Calif., (TaqMan® Universal Master Mix, cat. nos. 4304437, 4318157, and4326708).

Labeling Strategies

Any suitable labeling strategy can be employed in the methods describedherein. Where the reaction is analyzed for presence of a singleamplification product, a universal detection probe can be employed inthe amplification mixture. In particular embodiments, real-time PCRdetection can be carried out using a universal qPCR probe. Suitableuniversal qPCR probes include double-stranded DNA dyes, such as SYBRGreen, Pico Green (Molecular Probes, Inc., Eugene, Oreg.), Eva Green(Biotinum), ethidium bromide, and the like (see Zhu et al., 1994, Anal.Chem. 66:1941-48).

In some embodiments, one or more target-specific qPCR probes (i.e.,specific for a target nucleotide sequence to be detected) is employed inthe amplification mixtures to detect amplification products. Byjudicious choice of labels, analyses can be conducted in which thedifferent labels are excited and/or detected at different wavelengths ina single reaction (“multiplex detection”). See, e.g., FluorescenceSpectroscopy (Pesce et al., Eds.) Marcel Dekker, New York, (1971); Whiteet al., Fluorescence Analysis: A Practical Approach, Marcel Dekker, NewYork, (1970); Berlman, Handbook of Fluorescence Spectra of AromaticMolecules, 2nd ed., Academic Press, New York, (1971); Griffiths, Colourand Constitution of Organic Molecules, Academic Press, New York, (1976);Indicators (Bishop, Ed.). Pergamon Press, Oxford, 19723; and Haugland,Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes,Eugene (1992).

In some embodiments, it may be convenient to include labels on one ormore of the primers employed in in amplification mixture.

Exemplary Automation and Systems

In some embodiments, a target nucleic acid is detected using anautomated sample handling and/or analysis platform. In some embodiments,commercially available automated analysis platforms are utilized. Forexample, in some embodiments, the GeneXpert® system (Cepheid, Sunnyvale,Calif.) is utilized.

The methods described herein are illustrated for use with the GeneXpertsystem. Exemplary sample preparation and analysis methods are describedbelow. However, the present invention is not limited to a particulardetection method or analysis platform. One of skill in the artrecognizes that any number of platforms and methods may be utilized.

The GeneXpert® utilizes a self-contained, single use cartridge. Sampleextraction, amplification, and detection may all be carried out withinthis self-contained “laboratory in a cartridge.” (See e.g., U.S. Pat.Nos. 5,958,349, 6,403,037, 6,440,725, 6,783,736, 6,818,185; each ofwhich is herein incorporated by reference in its entirety for thisdescription.)

Components of the cartridge include, but are not limited to, processingchambers containing reagents, filters, and capture technologies usefulto extract, purify, and amplify target nucleic acids. A valve enablesfluid transfer from chamber to chamber and contains nucleic acids lysisand filtration components. An optical window enables real-time opticaldetection. A reaction tube enables very rapid thermal cycling.

In some embodiments, the GenXpert® system includes a plurality ofmodules for scalability. Each module includes a plurality of cartridges,along with sample handling and analysis components.

After the sample is added to the cartridge, the sample is contacted withlysis buffer and released nucleic acid is bound to a nucleicacid-binding substrate such as a silica or glass substrate. The samplesupernatant is then removed and the nucleic acid eluted in an elutionbuffer such as a Tris/EDTA buffer. The eluate may then be processed inthe cartridge to detect target genes as described herein. In someembodiments, the eluate is used to reconstitute at least some of thereagents, which are present in the cartridge as lyophilized particles.

In some embodiments, PCR is used to amplify and detect the presence ofone or more target nucleic acids. In some embodiments, the PCR uses Taqpolymerase with hot start function, such as AptaTaq (Roche).

In some embodiments, an off-line centrifugation is used to improve assayresults with samples with low cellular content. The sample, with orwithout the buffer added, is centrifuged and the supernatant removed.The pellet is then resuspended in a smaller volume of supernatant,buffer, or other liquid. The resuspended pellet is then added to aGeneXpert® cartridge as previously described.

Kits

Also contemplated is a kit for carrying out the methods describedherein. Such kits include one or more reagents useful for practicing anyof these methods. A kit generally includes a package with one or morecontainers holding the reagents, as one or more separate compositionsor, optionally, as an admixture where the compatibility of the reagentswill allow. The kit can also include other material(s) that may bedesirable from a user standpoint, such as a buffer(s), a diluent(s), astandard(s), and/or any other material useful in sample processing,washing, or conducting any other step of the assay.

Kits preferably include instructions for carrying out one or more of thescreening methods described herein. Instructions included in kits can beaffixed to packaging material or can be included as a package insert.While the instructions are typically written or printed materials theyare not limited to such. Any medium capable of storing such instructionsand communicating them to an end user can be employed. Such mediainclude, but are not limited to, electronic storage media (e.g.,magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM),and the like. As used herein, the term “instructions” can include theaddress of an internet site that provides the instructions.

EXAMPLES Example 1 Confirmation of Effect of “Clamp” Oligo onPrimer/target Structure

An experiment was performed in which the Tm was measured of a primeroligo (although called a “primer,” it was not used as such in thisexperiment) and a complimentary target sequence with and without the“clamp” oligo present. The target oligo was synthesized with a 5′fluorescent tag (fluorescein), and the primer incorporated afluorescence quenching moiety (see FIG. 6). The c sequence includesindicates the presence of a 2′ O-methyl backbone. The oligo sequencestested are listed in Table 4 below. The Tm of the right hand-mostdouble-helical region shown in the structures of FIG. 6 was measured byfollowing the increase in fluorescence that results as temperature isincreased and as the fluor and quencher are separated by melting of thisdouble-helical region.

If the region of primer to target binding were, as indicated in FIG. 6A,limited by the clamp to a b/b′ binding, then the Tm of that region wouldbe predicted to be much lower than in the situation in FIG. 6B withab/a′b′ binding. In Table 4 below are listed the oligos used in this andthe following experiment. In Table 5 are the predicted and observed Tm'sfor primer and target oligos, in the presence or absence of a clampoligo.

TABLE 4 Oligonucleotides used Oligo Cate- no. Sequence gory 161405′ggcgcuccggaccggcgTAGGCTGGTAACCAA primer CCGCTGAAGGCA(U01)ACGG3′(note: lower case = 2′-O-methyl;   U01 = dabcyl quencher-labeled uracil) (SEQ ID NO: 1) 16141 ggcgcuccggaccggcgTAGGCTGGTAACCAACC primerGCTGAAGGCA(U01)A-3′ (SEQ ID NO: 2) 161425′TGGTTACCAGCCTACGCCGGTCCGGAGCGCC3′ clamp block* (SEQ ID NO: 3) 161455′Fluorescein- target CCGTATGCCTTCAGCGGTTGGTTACCAGCCTACG CATT3′(SEQ ID NO: 4) 16146 5′Fluorescein-TATGC targetCTTCAGCGGTTGGTTACCAGCCTACGCATT3′ (SEQ ID NO: 5) 161475′CGTAGGCTGGTAACC3′ flank-  (SEQ ID NO: 6) ing primer 161485′GCGTAGGCTGGTAACC3′ flank-  (SEQ ID NO: 7) ing primer 161495′GCGT(A01)GGCTGGT(A01)ACC3′ flank-  (A01 = 2-aminopurine) ing(SEQ ID NO: 8) primer *block = moiety that blocks extension

TABLE 5 T_(m) measurements Predicted Tm Observed Tm Primer Target Clamp(deg. C.) (deg. C.) 16140 16145 None 76.4 (ab/a′b′) 78.5 16141 16146None 74.6 (ab/a′b′) 78.0 16140 16145 16142 68.3 (b/b′) 67.0 16141 1614616142 62.0 (b/b′) 66.5

The conditions for all hybrid melt analysis were: 0.01 M tris-HCl, 0.05M KCl and 0.006 M MgCl₂. All oligonucleotides were at 1 micromolar. Theoligo mixtures in Table 4 above were heated to 95 deg. C. and cooledslowly to 45 deg. C. and fluorescein fluorescence monitored using theCepheid SmartCycler™. The Tm was determined as temperature at which therate of fluorescence change was maximal.

The Tm's of b/b′ and ab/a′b′ were predicted using software(www.idtdna.com/analyzer/Applications/OligoAnalyzer). The observed Tm'sare consistent with a structure in which the region d′-a′ in the target,in the presence of a clamp oligo, remains single-stranded and availablefor hybridization.

The presence of a flanking primer (16147 to 16149) also at 1 micromolar,as diagrammed in FIG. 7A, made little difference in measured measuredTm's.

Example II Extensibility of Outer (Flanking) Primer

The extensibility of the outer (flanking) primer shown schematically inFIG. 7A was tested under the conditions shown in Table 6 in a PCRreaction.

TABLE 6 Reaction Flanking primer 1 16147 2 16148 3 16149 4 none

All reactions contained 10 mM Tris-HCl, 0.125 mM each dATP, dTTP, dCTPand dGTP, 0.15 micromolar of primer oligo 16140 from table 1, above,0.125 micromolar of target oligo 16145 from table 1, 0.125 micromolar ofclamp oligo 16142 from table 1, 45 mM KCl, 3.5 mM MgCl₂, 14 units ofAmpliTaqCS, which has DNA polymerase activity but neither 5′ to 3′ nor3′ to '5 exonuclease activity, and 15 units of antibody to Taqpolymerase, which provides a temperature activated “hot-start” to theincorporation reaction.

0.125 mM or no flanking primer was added as per Table 6 above; reactionswere monitored over time using the SmartCycler while raising thetemperature to 95 degrees to separate the oligos and simultaneouslyactivate the polymerase, then lowering the temperature to 60 degrees toallow the oligos to anneal and to allow any primer extension to occur.The results are shown in 6B. The three rising traces are separatereactions with slightly different clamps; the flat trace is without theouter (flanking), displacing primer present. These results indicate thata strand displacing reaction that displaces the quencher occurs when theouter (flanking) primer is present.

What is claimed is:
 1. A nucleic acid primer set for amplifying a targetnucleic acid in a sample, wherein the target nucleic acid comprises afirst template strand and, optionally, a second template strand, whereinthe second template strand is complementary to the first templatestrand, the primer set comprising oligonucleotides in the form of, orcapable of forming, at least two first primers capable of hybridizing tothe first template strand, wherein the at least two first primerscomprise a first outer primer and a first inner primer, the first outerprimer comprising a primer sequence a that specifically hybridizes tofirst template strand sequence a′; and the first inner primer comprisinga single-stranded primer sequence b that specifically hybridizes tofirst template strand sequence b′, wherein b′ is adjacent to, and 5′ of,a′, and wherein single-stranded primer sequence b is linked at its 5′end to a first strand of a double-stranded primer sequence comprising: aprimer sequence a adjacent to, and 5′ of, single-stranded primersequence b; and a clamp sequence c adjacent to, and 5′ of, primersequence a, wherein clamp sequence c is not complementary to a firststrand template sequence d′, which is adjacent to, and 3′ of, firststrand template sequence a′, wherein the double-stranded portion of theinner primer comprises combined sequence c-a annealed to a complementarycombined sequence a′-c′; wherein sequence c comprises 2′-O-methyl RNAand/or combined sequence a′-c′ cannot be extended from its 3′ end; andwherein combined sequence c-a, in double-stranded form, is more stablethan combined sequence a-b, in double-stranded form.
 2. The primer setof claim 1, wherein the primer set additionally comprises at least onesecond primer capable of specifically hybridizing to the second templatestrand.
 3. A method for amplifying a target nucleic acid in a sample,wherein the target nucleic acid comprises a first template strand and,optionally, a second template strand, wherein the second template strandis complementary to the first template strand, the method comprising:(a) contacting the sample with: (i) at least two first primers capableof hybridizing to the first template strand, wherein the at least twofirst primers comprise a first outer primer and a first inner primer,the first outer primer comprising a primer sequence a that specificallyhybridizes to first template strand sequence a′; and the first innerprimer comprising a single-stranded primer sequence b that specificallyhybridizes to first template strand sequence b′, wherein b′ is adjacentto, and 5′ of, a′, and wherein single-stranded primer sequence b islinked at its 5′ end to a first strand of a double-stranded primersequence comprising: a primer sequence a adjacent to, and 5′ of,single-stranded primer sequence b; and a clamp sequence c adjacent to,and 5′ of, primer sequence a, wherein clamp sequence c is notcomplementary to a first strand template sequence d′, which is adjacentto, and 3′ of, first strand template sequence a′, wherein thedouble-stranded portion of the inner primer comprises combined sequencec-a annealed to a complementary combined sequence a′-c′; whereinsequence c comprises 2′-O-methyl RNA and/or combined sequence a′-c′cannot be extended from its 3′ end; and wherein combined sequence c-a,in double-stranded form, is more stable than combined sequence a-b, indouble-stranded form; and (ii) at least one second primer capable ofspecifically hybridizing to the second template strand, wherein thecontacting is carried out under conditions wherein the primers anneal totheir template strands, if present; and (b) amplifying the targetnucleic acid, if present, using a DNA polymerase lacking 5′-3′exonuclease activity, under conditions where strand displacement occurs,to produce amplicons that comprise sequence extending from templatesequence a′ to the binding site for the second primer.
 4. The primer setof claim 1, wherein the DNA polymerase comprises strand displacementactivity.
 5. The primer set of claim 1, wherein the T_(m) of combinedsequence c-a, in double-stranded form, is greater than that of combinedsequence a-b, in double stranded form.
 6. The primer set of claim 1,wherein combined sequence c-a is more GC-rich than combined sequencea-b, and/or contains more stabilizing bases.
 7. The method of claim 3,wherein said amplifying amplifies the target nucleic acid at the rate ofup to 3^(number of cycles) during the exponential phase of PCR.
 8. Themethod of claim 3, wherein said amplifying permits detection of a singlecopy nucleic acid in a biological sample within about 12%-42% feweramplification cycles than would be required for said detection usingonly a single forward and a single reverse primer.
 9. The primer set ofclaim 1, wherein the second primer comprises oligonucleotides in theform of, or capable of forming, at least two second primers capable ofhybridizing to the second template strand, wherein the at least twosecond primers comprise a second outer primer and a second inner primer,the second outer primer comprising a primer sequence e that specificallyhybridizes to second template strand sequence e′; and the second innerprimer comprising a single-stranded primer sequence f that specificallyhybridizes to second template strand sequence f′, wherein f′ is adjacentto, and 5′ of, e′, and wherein single-stranded primer sequence f islinked at its 5′ end to a first strand of a double-stranded primersequence comprising: a primer sequence e adjacent to, and 5′ of,single-stranded primer sequence f; and a clamp sequence g adjacent to,and 5- of, primer sequence e, wherein clamp sequence g is notcomplementary to second strand template sequence h′, which is adjacentto, and 3′, of second strand template sequence e′.
 10. The primer set ofclaim 9, wherein the T_(m) of combined sequence g-e, in double-strandedform is greater than that of combined sequence e-f, in double-strandedform.
 11. The primer set of claim 9, wherein combined sequence g-e ismore GC-rich than combined sequence e-f, and/or contains morestabilizing bases.
 12. The primer set of claim 9, wherein saidamplifying amplifies the target nucleic acid at the rate of up to6^(number of cycles) during the exponential phase of PCR.
 13. The primerset of claim 9, wherein said amplifying permits detection of a singlecopy nucleic acid in a biological sample within about 36%-66% feweramplification cycles than would be required for said detection usingonly a single forward and a single reverse primer.
 14. The primer set ofclaim 9, wherein clamp sequence g is not capable of being copied duringamplification.
 15. The primer set of claim 14, wherein clamp sequence gcomprise(s) 2′-O-methyl RNA.
 16. The primer set of claim 1, wherein thedouble-stranded primer sequence of the first inner primer does notcomprise a hairpin sequence.
 17. The primer set of claim 1, wherein thedouble-stranded primer sequence of the first inner primer comprises ahairpin sequence in which clamp sequence c is linked to complementarysequence c′.
 18. A nucleic acid primer set for amplifying a targetnucleic acid in a sample, wherein the target nucleic acid comprises afirst template strand and, optionally, a second template strand, whereinthe second template strand is complementary to the first templatestrand, the primer set comprising oligonucleotides in the form of, orcapable of forming, at least three first primers capable of hybridizingto the first template strand, wherein the at least three first primerscomprise a first outer primer, a first intermediate primer, and a firstinner primer, the first outer primer comprising a primer sequence d thatspecifically hybridizes to first template strand sequence d′; the firstintermediate primer comprising a primer sequence a that specificallyhybridizes to first template strand sequence a′, wherein a′ is adjacentto, and 5′ of, d′, and wherein single-stranded primer sequence a islinked at its 5′ end to a first strand of a double-stranded primersequence comprising: a primer sequence d adjacent to, and 5′ of,single-stranded primer sequence a; and a clamp sequence c1 adjacent to,and 5- of, primer sequence d, wherein clamp sequence c1 is notcomplementary to a first strand template sequence i′, which is adjacentto, and 3′ of, first strand template sequence d′, wherein thedouble-stranded portion of the first intermediate primer comprisescombined sequence c1-d annealed to a complementary combined sequenced′-c1′; wherein sequence c1 comprises 2′-O-methyl RNA and/or combinedsequence d′-c1′ cannot be extended from its 3′ end; and wherein combinedsequence c1-d, in double-stranded form, is more stable than combinedsequence d-a, in double-stranded form; and the first inner primercomprising a single-stranded primer sequence b that specificallyhybridizes to first template strand sequence b′, wherein b′ is adjacentto, and 5′ of, a′, and wherein single-stranded primer sequence b islinked at its 5′ end to a first strand of a double-stranded primersequence comprising: a primer sequence a adjacent to, and 5′ of,single-stranded primer sequence b; a primer sequence d adjacent to, and5′ of, primer sequence a; and a clamp sequence c2 adjacent to, and 5′of, primer sequence d, wherein clamp sequence c2 is not complementary tofirst strand template sequence i′, wherein the double-stranded portionof the first inner primer comprises combined sequence c2-d-a annealed toa complementary combined sequence a′-d′-c2′; wherein sequence c2comprises 2′-O-methyl RNA and/or combined sequence a′-d′-c2′ cannot beextended from its 3′ end; and wherein combined sequence c2-d-a, indouble-stranded form, is more stable than combined sequence d-a-b, indouble-stranded form.
 19. A method for amplifying a target nucleic acidin a sample, wherein the target nucleic acid comprises a first templatestrand and, optionally, a second template strand, wherein the secondtemplate strand is complementary to the first template strand, themethod comprising: (a) contacting the sample with: (i) at least threefirst primers capable of hybridizing to the first template strand,wherein the at least three first primers comprise a first outer primer,a first intermediate primer, and a first inner primer, the first outerprimer comprising a primer sequence d that specifically hybridizes tofirst template strand sequence d′; the first intermediate primercomprising a primer sequence a that specifically hybridizes to firsttemplate strand sequence a′, wherein a′ is adjacent to, and 5′ of, d′,and wherein single-stranded primer sequence a is linked at its 5′ end toa first strand of a double-stranded primer sequence comprising: a primersequence d adjacent to, and 5′ of, single-stranded primer sequence a;and a clamp sequence c1 adjacent to, and 5- of, primer sequence d,wherein clamp sequence c1 is not complementary to a first strandtemplate sequence i′, which is adjacent to, and 3′ of, first strandtemplate sequence d′, wherein the double-stranded portion of the firstintermediate primer comprises combined sequence c1-d annealed to acomplementary combined sequence d′-c1′; wherein sequence c1 comprises2′-O-methyl RNA and/or combined sequence d′-c1′ cannot be extended fromits 3′ end; and wherein combined sequence c1-d, in double-stranded form,is more stable than combined sequence d-a, in double-stranded form; andthe first inner primer comprising a single-stranded primer sequence bthat specifically hybridizes to first template strand sequence b′,wherein b′ is adjacent to, and 5′ of, a′, and wherein single-strandedprimer sequence b is linked at its 5′ end to a first strand of adouble-stranded primer sequence comprising: a primer sequence a adjacentto, and 5′ of, single-stranded primer sequence b; a primer sequence dadjacent to, and 5′ of, primer sequence a; and a clamp sequence c2adjacent to, and 5′ of, primer sequence d, wherein clamp sequence c2 isnot complementary to first strand template sequence i′, wherein thedouble-stranded portion of the first inner primer comprises combinedsequence c2-d-a annealed to a complementary combined sequence a′-d′-c2′;wherein sequence c2 comprises 2′-O-methyl RNA and/or combined sequencea′-d′-c2′ cannot be extended from its 3′ end; and wherein combinedsequence c2-d-a, in double-stranded form, is more stable than combinedsequence d-a-b, in double-stranded form; and (ii) at least one secondprimer capable of specifically hybridizing to the second templatestrand, wherein the contacting is carried out under conditions whereinthe primers anneal to their template strands, if present; and (b)amplifying the target nucleic acid, if present, using a DNA polymeraselacking 5′-3′ exonuclease activity, under conditions where stranddisplacement occurs, to produce amplicons that comprise sequenceextending from template sequence a′ to the binding site for the secondprimer.